t^be mnlversit^ ot Cbtcaao 



DECAY AND SOIL TOXINS 



A DISSERTATION 

SUBMITTED TO THE FACULTY OF THE OGDEN GRADUATE SCHOOL 

OF SCIENCE IN CANDIDACY FOR THE DEGREE 

OF DOCTOR OF PHILOSOPHY 

(department of botany) 



BY 



GEORGE B. RIGG 



A Private Edition 

Distributed By 

The University of Chicago Libraries 



Reprinted from 

The Botanical Gazette, Vol. LXI, No. 4 

Chicago, igi6 



Zhc mniversit^ of CbicaQO 



DECAY AND SOIL TOXINS 



A DISSERTATION 

SUBMITTED TO THE FACULTY OF THE OGDEN GRADUATE SCHOOL 

OF SCIENCE IN CANDIDACY FOR THE DEGREE 

OF DOCTOR OF PHILOSOPHY 

(department of botany) 



GEORGE B. RIGG 



A Private Edition 

Distributed By 

The University of Chicago Libraries 



Reprinted from 

The Botanical Gazette, Vol. LXI, No. 4 

Chicago, IQ16 



5 6^*^ 



6 



Digitized by the Internet Archive 
in 2010 \A?Wi^ftrffdrhg from 
The Library 6t Congress 



http://www.archive.org/details/decaysoiltoxinsOOrigg 



Reprinted from the Botanical Gazette, 6i: No. 4, April 1916 



DECAY AND SOIL TOXINS 

CONTRIBUTIONS FROM THE HULL BOTANICAL LABORATORY 213 

George B. Rigg 

The decomposition products of a specific plant organ and their 
effects on the growth of other plants furnished the point of attack 
for the work on toxicity reported in this paper. The material 
used was the rhizomes of Nymphaea advena Ait. and A^. polysepala 
Greene. This material was obtained at intervals from July 191 2 
to October 191 5, at various places in the vicinity of Chicago, 
Illinois, and Seattle, Washington. 

Review of literature 

RELATED WORK ON TOXICITY 

The organic constituents of soils have been under investigation 
by workers in the United States Bureau of Soils for 10 years. 
Livingston (12, 13) found toxic substances, probably organic, 
in an unproductive soil. Schreiner (21) and his co-workers have 
isolated from soils more than 25 organic compounds differing widely 
in chemical character. Some of these (for example, dihydroxy- 
stearic acid) have proved harmful to growing plants; some (for 
example, nucleic acid) have been found beneficial; and some have 
not been shown to have any effect on the growth of plants. Bot- 
TOMLY (3) has found that certain aerobic organisms grow well 
in peat and form from it compounds that are beneficial to the growth 
of plants. He suggests that very small amounts of accessory 
organic substances may be necessary for the growth of plants. 

Humic acid has been much discussed as a possible factor in 
plant growth. Not only the effects of this so-called humic acid, 
but also the constitution and nature of the substance are in doubt. 
Schreiner (21) regards it as a mixture of substances. Wieler (29) 
takes the view that humic acids in soils are inorganic acids result- 
ing, for example, from the chemical decomposition of salts. 

Bauman and Gully (2) have suggested that the acidity of bog 
water is due to the fact that the cell colloids of the disintegrating 
295] [Botanical Gazette, voL 61 



296 BOTANICAL GAZETTE [april 

plant tissues retain chiefly the basic ions of the salts dissolved 
in the cell contents of the plant tissues before they began to decay, 
thus freeing the acid ions. Skene (24) has found that various 
species of Sphagnum thrive best in acid solution because mineral 
solutions, although usually physiologically harmless, may be 
ecologically harmful. 

Work by Livingston (14), Dachnowski (6, 7), the writer (19), 
and others (20), indicates that the inhibition from sphagnum bogs 
of plants other than bog xerophytes is not due to acidity, or to 
low surface tension, or to high osmotic pressure of the soil solu- 
tion, but is due in part to the presence of toxic substance or 
substances in the soil solution. 

Many workers (8, 9, 15-18, 25-27) have found that cultivated 
crops and plants grown in cultures have a favorable or an unfavor- 
able influence on other plants growing in the same substratum 
either at the same time or subsequently. Food supply and toxins 
have been suggested as means through which this influence may 
be exerted. Czapek (5) finds that the roots of plants are 
injured when the surface tension of the bathing solution is lower 
than 0.66. 

Sherff (23) found in Skokie marsh near Chicago that where the 
rhizomes of Sagittaria latifolia had penetrated the decaying rhizomes 
of Nymphaea advena, they themselves had begun to decay. 

sterile cultures of seed plants 

More or less success has been attained by various workers in 
attempts to -grow seed plants under sterile conditions. Harrison 
and Barlow (ii) tried sterilization by dry heat, moist heat, 
sulphuric acid, calcium hydrate, formaldehyde, and mercuric 
chloride, and abandoned all of these means. They succeeded in 
getting sterile cultures of certain legumes by treating the unopened 
pods with mercuric chloride, opening them with flamed forceps, 
and transferring the seeds to a very small quantity of boiling water 
in sterile test tubes. 

Wilson and Harding (30) tried alcohol, formaldehyde, and 
mercuric chloride as a means of steriHzing alfalfa seeds, but found 
that when the seeds were sterile, the germination was very low. 



1^16] RIGG—SOIL TOXINS 297 

Using a modification of Harrison and Barlow's method, they 
got alfalfa seedlings which grew in sterile cultures for 4 months. 

Brown (4) found that barley seeds take up water from a 
fairly strong solution of sulphuric acid and remain uninjured. 
ScHROEDER (22) found silver nitrate to be a good means of steriliz- 
ing wheat. He found that hand-picked wheat endured soaking 
in a 5 per cent solution of this substance for 24 hours without 
injury, and for 72 hours with but slight injury. Threshed wheat, 
however, because of the rupture of semipermeable membranes by 
the machinery, would not stand such prolonged treatment. Archi- 
CHOWSKY (i) got a large percentage of sterile cultures of seed 
plants by the use of formaldehyde and other antiseptic agents on 
peas, pumpkins, and other seeds. 

Solutions and preparations 

Both of the species of Nymphaea used produce branched 
rhizomes 3-15 cm. thick and sometimes reaching as great a length 
as 3 m., although they are more commonly i m. or less. The 
older portions of these rhizomes decay. Sherff (23) found these 
rhizomes decaying to within a short distance of the growing apex. 
The writer has found the decay only in older portions of the rhizome. 
Sound pieces of the rhizome were collected and the following 
solutions were made up quantitatively, each solution having a 
volume of 1600 cc. and containing the solutes obtained by the 
methods described from 1000 gm. of fresh rhizome. An average 
of 3 tests on the water contents of the fresh rhizome gives 88 per 
cent of water. The tests were made by cutting 500 gm. of the 
fresh material into small pieces and drying it at 105° C. 

Solution jyl.— This was the hquid resulting from the decay 
of 1000 gm. of fresh Nymphaea rhizome in redistilled water, freed 
from sohd matter by filtering through cheesecloth, and diluted 
to 1600 cc. with redistilled water. Molds continued to grow 
on the surface of this solution. It was amber colored. 

Solution I B.— This was the solution remaining after a duplicate 
of I A had been extracted by shaking with an equal volume of 
ether in a separatory funnel. The ether that dissolved in the 
water solution was removed by heating to 40° C. and subjecting to 



298 BOTANICAL GAZETTE [april 

a suction of 2 cm. of mercury with an aspirator. Molds continued 
to grow on the surface of this solution. It had a slightly darker 
color than lA. 

Solution iC. — The ether used in extracting iB was allowed to 
evaporate spontaneously. The solid remaining was reddish- 
brown, and only partially soluble in water. This solution consists 
of 1600 cc. of redistilled water, and all the residue from the ether 
extraction that would dissolve in that quantity of water at 40° C. 
No molds grew on this solution, and no scum or turbidity or other 
evidence of bacterial activity appeared. 

Preparation iD. — This was the solid remaining from filtering 
lA. This solid was ground with an equal volume of sand. 

Solution 2A. — This was the distillate under reduced pressure 
(2 cm. pressure), at 40° C, of the Hquid and sohd products of the 
decay of 1000 gm. of Nymphaea rhizome in redistilled water. This 
was a clear liquid having the appearance of water. No molds 
grew upon it, and it showed no evidence of bacterial activity. 

Solution 2B. — The solid remaining from the distillation of 
2 A was dried in an oven at 30° C. and then ground in a mortar. 
It was then black powder. This was extracted in a Soxhlet appa- 
ratus with ether. When the ether was allowed to evaporate spon- 
taneously, a sticky, semi-sohd, reddish-yellow substance remained. 
This was only partially soluble in water. This solution represents 
1600 cc. of redistilled water, with all of the ether extract that would 
dissolve in it at 40° C. It had a hght reddish-yellow color. No 
molds grew upon it, and no evidence of bacterial activity appeared. 

Solution 2C — The solid remaining from the extract of 2B 
was exposed to air until all odor of ether had disappeared. It 
was then extracted for 2 hours with 1600 cc. of redistilled water. 
It was perfectly clear. No molds or evidence of bacterial growth 
appeared. 

Preparation 2D. — This represents the sohd remaining from the 
extract of 2C, ground in a mortar with an equal volume of sand 
to form a soil. 

Solution 3 A . — One kg. of fresh Nymphaea rhizome was cut into 
pieces, ground in a meat grinder, and the juice pressed out in a 
fruit press. The solid remaining was extracted with ether, and 



1916I RIGG—SOIL TOXINS 299 

afterward exposed to the air until the odor of ether disappeared. 
It was then allowed to decay in redistilled water. The Hquid 
resulting from this decay was strained through cheesecloth and 
diluted with redistilled water to 1600 cc. 

Solution jB. — The juice squeezed out in preparing solution 
4A was extracted by shaking with an equal volume of ether in a 
separatory funnel, which was then freed from ether by heating 
to 40° C, and subjecting to suction (2 cm. of mercury) by means 
of an aspirator for 24 hours. This, when diluted 1600 cc, consti- 
tuted solution 3B. 

Solution jC. — The ether used in extracting 3A was combined 
with that used in extracting 3B. This was allowed to evaporate 
spontaneously and as much of the residue as possible was taken 
up in 1600 cc. of redistilled water at 40° C. 

Preparation jD. — The solid matter remaining on the cheese- 
cloth in the preparation of 3A was air-dried and ground with an 
equal volume of sand to form a soil. 

Solution 4A. — This was the water extract of the fresh rhizome 
made under sterile conditions. The rhizome was cut into small 
pieces and placed in flasks with water. These flasks were stoppered 
with cotton and sterilized in an autoclave. This solution stood 
sterile for 1 1 months before its toxicity was tested. 

Solution 5 A . — This was the water solution of the ash from the 
fresh rhizome. 

All sand used in the previous preparations and in the follow- 
ing experiments was either no. 2^ quartz or "Ottawa test." In 
all cases it was washed in 10 per cent HCl, freed from acid by 
washing in running water, and finally rinsed with redistilled water. 

The "Knop's solution" had the following composition: i part 
KNO3; I part K.HPO4; I part Mg SO4; 4 parts CaCNOj)^. This 
was made up to o . i per cent. 

Where "tap water" is mentioned, the water used was Chicago 
city water. Where "Cedar River water" is mentioned, the water 
used was Seattle city water, which is piped from the river near 
its origin in a snow-fed lake. Where "Lake Washington water" 
is mentioned, the water is that supplied from Lake Washington 
to the botany laboratories at the University of Washington. 



300 



BOTANICAL GAZETTE 



[APRIL 



Experiments and results 

In order to determine the relative toxicity of these various 
solutions to Tradescaniia, cuttings of the plant were placed in 
various dilutions of each solution with redistilled water. In this 
way the percentage of the solution (that is, the number of cc. 
diluted to loo with redistilled water) that would allow the forma- 
tion of roots but inhibit the production of root hairs was determined. 

All of the solutions except 5 A were acid to both litmus and 
phenolphthalein. Their acidity was determined by titrating with 
N/io NaOH, using phenolphthalein as an indicator. 

Table I gives the toxic limits of these solutions to Tradescantia 
(as previously defined) and their acidity, together with the relative 
rank of each solution as to toxicity and acidity and the ratios of 
these. 

TABLE I 



Solution acidity 


Toxicity 
(per cent) 


Rank as to 
acidity 


Rank as to 
toxicity 


Ratio of acidity 
to toxicity 


lA N25/1000 

iB N24/1000 


7-5 

7-S 

7-5 

10. 

50.0 

12. S 


I 
2 

4 
3 
7 
5 
6 
8 


I, 2, or 3 

I, 2, or 3* 

I, 2, or 3 

4 

7 
5 
6 
8 


0.27 
0.25 
0. 12 
0. 24 
0. 12 
0. ID 
0.08 
0.06 


iC Nii/iooo 

2A N22/1000 


2B N6 /looo 

3A Ng /looo 

3B N7 /looo. . . ." ; 

3C N5 /lOOO 



All of the solutions mentioned in table I, except 3B, were 
neutralized to phenolphthalein with N/io sodium hydrate, and 
the effect of both the acid and the neutral solution was tried on 
Tradescantia cuttings. Table II shows the results of the dilutions 
named on Tradescantia cuttings, 

TABLE II 



Solution 


Strength 
(per cent) 


Acid 


Neutralized 


lA 

iB 

iC 

2A 

2B 

3A 


7 

5 

5 

7 

60 

12 

10 


5 



5 


S 



Root hairs none 

" " slightly stunted 

" " none 
Roots none 
Root hairs none 

" " slightly stunted 


Root hairs normal 

" " normal 

" slightly stunted 
Roots 3-10 mm. long 
Root hairs slightly stunted 

" " normal 


3C 











iQi6] RIGG—SOIL TOXINS 301 

The toxicity of the first 4 of these solutions when undiluted 
was not perceptibly lowered by neutralization with sodium hydrate. 

A 20 per cent solution of lA was shaken in a large flask 5 times 
a day for 10 minutes each time, for 3 days in order to aerate it, 
and then filtered through a filter paper. Tests on Tradescantia 
indicated that its toxicity was not decreased by this treatment. 

A 20 per cent solution of solution A in tap water was shaken 
with animal charcoal and filtered. The filtrate was colorless and 
odorless. The solution before this treatment had an amber color 
and a foul odor. Cuttings of Tradescantia were placed in this. In 
all cases they grew well and produced good roots with normal or 
only slightly stunted root hairs. 

The animal charcoal with which this solution was shaken was 
used as a soil for cultures of alfalfa seed. Controls of animal 
charcoal shaken with tap water were run. The growth in the 
controls was twice als great as the growth in the cultures. 

The toxicity of preparation 2D to Tradescantia cuttings was 
tested by planting the cuttings in the preparation in small flower 
pots. This preparation was further diluted also by grinding with 
half its volume of sand. For convenience this further dilution is 
referred to in table III as preparation 2Dx. Controls were run 

TABLE III 



Medium 


Roots 


Plants 


2D 


None 

Normal 

Normal 


Dying 


2Dx 


Healthy 


Potting soil 


Healthy 



in potting soil. All of the pots in a set were placed together in a 
large glass dish, so that they stood in about one-fourth of an inch 
of tap water and thus were all watered aHke. Table III shows the 
results at the end of 14 days. 

Preparation 3D was tried in the same way with similar results. 
Preparation iD was tried in the same way with corn and did not 
prove toxic. Preparations 2D and 3D are not at all toxic to alfalfa. 

The toxicity of solutions lA, iB, iC, and 2A to unsterilized 
corn was tested by planting the seeds in 200 cc. of sand watered 
with 25 cc. of solution. The cultures were in 500 cc. Erlenmeyer 



302 BOTANICAL GAZETTE [apeil 

flasks, stoppered with cotton. Controls watered with Knop's 
solution were run. All of the solutions tested proved toxic, but 
2A was found to be less toxic to corn under these conditions than 
the others. The toxicity of the solutions was shown in the injury 
to the root tips, causing a great decrease in the total length of root 
produced as compared with the controls, and thus eventually 
kilhng the plants. 

The efTect of the 8 solutions listed in table I was tried on un- 
sterilized alfalfa in sand. They were all found extremely toxic, 
many of the plants dying as they emerged from the sand, and the 
best of them attaining only one-fifth of the height attained by 
controls watered with tap water. 

It was found by tests that tomato seeds did not germinate at 
all in solution lA in sand, while controls in sand watered with tap 
water grew vigorously. 

An attempt was made to secure sterile cultures of corn in 500 cc. 
Erlenmeyer flasks in order to determine whether the presence of 
organisms in the cultures was a factor in the toxicity of these cul- 
tures. The sterility of the cultures was tested by making bouillon 
cultures from various portions of the sand and the seeds at the end 
of the experiment; 65 per cent of the cultures proved sterile. 

Three problems were to be solved in working out a method of 
securing sterile cultures of seed plants: (i) the sterilization of the 
flasks and contents; (2) the sterilization of the seeds; (3) the 
transfer of the sterile seeds to the sterile flasks under sterile 
conditions. 

The cotton-stoppered flasks, containing 200 cc. of sand and 
20 cc. of the solution, were steriHzed for i hour at 15 lbs. pressure 
in the autoclave. A solution of silver nitrate was used as a means 
of sterilizing the seeds. It was found by experiment that corn 
would germinate , well after treatment for i minute with N/300 
AgN03 and subsequent washing with water. As a means of mak- 
ing the transfer of the seeds to the flask, the box previously used by 
Jensen (iia) was used. When the lid of the box was closed, 
the operator could thrust his hands into the gloves and work 
without danger of contaminating the cultures from any source 
outside of the box. Since the entire top and part of the front of 



i9i6] RIGG—SOIL TOXINS 303 

the box were of glass, all articles inside of the box could be seen 
readily by the operator. 

The entire inside of the box, including the side of the gloves 
and sleeves exposed to the air of the box, and the outside of all 
flasks and other articles placed in the box, were treated thoroughly 
with a 15 per cent solution of glycerine saturated with carbolic 
acid. All of the cultures prepared were thus more or less exposed 
to carbolic acid fumes. This solution was applied by means of a 
sponge attached to a short stick. It was found that flasks and 
dishes stuck to the paint treated with this mixture, so that a false 
bottom of glass was placed in the box. The stoppers of the steri- 
lized flasks (including 2 of sterile water) were flamed, and the flasks 
placed in the box. Four tall stenders with ground glass covers 
were sterilized in an oven for 5 hours at 120° C, and placed in the 
box. A bottle of N/300 AgNoj was also placed in the box, as 
were also a pair of lo-inch brass forceps and a waste jar containing 
a little of the glycerine carbolic acid solution. Everything in the 
box having been treated with the antiseptic solution, the cover of 
one of the stenders was removed and dry corn placed in the stender 
and the cover quickly replaced. In selecting corn for this purpose, 
care had been taken to secure smooth kernels that would offer little 
opportunity for the lodgment of air bubbles in the dent or elsewhere. 

The box now remained closed over night, to allow any organisms 
in the air to settle to the bottom of the box and be held by the 
glycerine solution. In the morning the operator thrust his hands 
into the gloves, poured AgNOj on the seeds, left it on for i minute, 
poured it off into the waste jar, and then washed the seeds in several 
changes of sterile water, finally allowing them to soak in it for 
several hours, and rinsing them again. The tips of the forceps 
now were washed thoroughly in the sterile water and the transfer 
of the seeds was made. In some cultures the seeds were thrust 
down into the sand by means of these forceps, and in some cases 
they were left on the surface. After 2 or 3 cultures had been made, 
the forceps were placed in the antiseptic solution for a few minutes 
and again rinsed in sterile water. After the cultures were prepared, 
they were removed from the box and placed near a window in the 
laboratory. 



304 BOTANICAL GAZETTE [april 

The results showed that solutions lA, iB, iC, and 2A (these 
were the only ones tried) are just as toxic under these con- 
ditions as when organisms were known to be present in abundance. 
Since 65 per cent of these cultures were shown (by bouillon cultures 
from them at the close of the experiment). to be sterile, it is evident 
that the presence of organisms is not a necessary condition of the 
toxicity of these solutions. 

Solutions I A, iB, and iC likewise proved toxic to alfalfa in 
cultures prepared as previously described for corn. The same 
precautions for securing sterility were observed, but tests of sterility 
were not made. 

The toxicity of solution 4A to Tradescantia cuttings was tested 
as follows: wide-mouth 50 cc. bottles were filled with dilutions of 
the following strength: 20, 15, and 10 per cent. Into each of 
these was placed a cutting of Tradescantia. The mouths of the 
bottles were left open. At the end of 16 days no root hairs had 
formed on any of these plants and all of the plants were showing 
signs of death. The controls in Lake Washington water all had 
abundant root hairs and were in healthy condition. 

Other sets of bottles were prepared as previously described, 
except that the mouths were stoppered with cotton and the bottles 
containing the liquids were sterilized in the autoclave. The dilu- 
tions were as follows: undiluted, 20, 15, and 10 per cent. Each 
cotton stopper was then displaced just enough to allow a cutting 
of Tradescantia to be placed in the hquid. None of these plants 
developed normal root hairs. All of them except those in the 10 
per cent dilution showed signs of death at the end of 16 days. 
Turbidity due to the action of organisms was evident in all of these, 
and molds grew on some of them. 

Flasks (500 cc.) were prepared, cotton-stoppered, each con- 
taining 180 gm. of sand and 18 cc. of solution. These were auto- 
claved at 12 lbs. pressure for i hour. Solution 4A was used pure 
and also in the following solutions: 20, 15, and 10 per cent. Con- 
trols with tap water were also run. Corn was treated with N/ioo 
silver nitrate for 2 minutes, then with sterile forceps 5 kernels were 
placed in each flask. At the end of 16 days the growth was notice- 
ably greater in all of the controls than in any of the solutions. All 



i9i6] RIG&-SOIL TOXINS 305 

of the flasks except 2 (one control and one 15 per cent) had molds 
growing in them. Bouillon and also agar cultures were made from 
each of these, and the 15 per cent flask proved to be sterile. The 
other was not sterile. These 2 flasks were kept a week longer and 
the growth in the control was much better than that in the 15 
per cent solution. 

The transfers of the corn in these cases were made in the labora- 
tory, without the use of the sterile box just described. It is 
hoped that by autoclaving the flasks at a higher pressure, and treat- 
ing the seeds with the silver nitrate for a longer period, a larger 
number of sterile flasks could be obtained. 
■ Solution lA was filtered and the fresh filtrate was immediately 
saturated with ammonium sulphate. The sulphate was added 
gradually and the solution was shaken after each addition. No 
precipitate appeared at once, but when it had stood over night a 
considerable amount of a brownish precipitate was present. Some 
of the precipitate was at the surface of the liquid, some had settled 
to the bottom, and some particles were in suspension in the liquid. 

The precipitate was filtered off and redissolved in a volume 
of Cedar River water equal to the original volume of the solution. 
Both filtrate and precipitate were then dialyzed in dialyzing tubing 
in running water for 1 1 days. At the end of this time they showed 
no precipitate with barium chloride. The filtrate and the pre- 
cipitate were then tested for toxicity by placing Tradescantia 
cuttings in them. Root hairs were formed in both, but their 
development was poorer in the solution of the precipitate than 
in the filtrate. 

In preparing solution 5A, 2 . 83 gm. of ash were obtained from 
500 gm. of fresh rhizomes. This is 4. 7 per cent of the dry weight. 
Approximately half of this went into solution when shaken with 
800 cc. of Cedar River water at 18° C. The solution was basic 
to litmus. In all cases tried with 5 A and its dupHcates the toxicity 
to Tradescantia cuttings was so marked that practically no root 
hairs developed and the plants soon died. When dilutions were 
tried it was found that all dilutions down to 10 per cent (10 cc. 
solution to 90 cc. water) inhibited root hair production; in 5 per 
cent dilution root hairs developed normally. 



3o6 BOTANICAL GAZETTE [apeil 

A solution was prepared from each of the following substances, 

by allowing a quantity of it to decay in redistilled water: potato, 

turnip, rhizome of Castilia odorata, and rhizome of Typha latifolia. 

These solutions approximated the strength of solution lA prepared 

from Nymphaea. They all proved toxic to Tradescantia cuttings, 

but to a less degree than lA did. Their toxicity was in the order 

named. 

Discussion 

It is evident from the data given that even very dilute solu- 
tions of the products of the decay of Nymphaea rhizomes are toxic 
to Tradescantia cuttings in water cultures and the seedlings of 
tomato, alfalfa, and corn in sand cultures. 

Although the products of the decay of the subterranean parts 
of other plants proved toxic, the toxicity of the products of the 
.decay of Nymphaea rhizomes was considerably greater than that 
of any other plant parts experimented on. While it is possible 
that toxicity from decay is rather common, Nymphaea seems to 
merit particular attention in this regard. The dilution of solution 
I A that entirely inhibited the formation of root hairs on Trades- 
cantia cuttings contained in each cc. the products of the decay of 
4.7mg. of fresh rhizome. Since only 12 per cent of the fresh 
rhizome is soHd matter, the amount of sohd whose decay con- 
tributed to the solutions in each cc. of the toxic solution was 
o.56mg. 

The fact that the solutions listed in table I were all acid, and 
that their toxicity was largely destroyed by neutrahzation with 
sodium hydrate, would seem to suggest acidity as a large factor 
in the toxicity. The toxicity is not proportional to the acidity 
as determined by the titration method. It may be proportional, 
however, to the H ion concentration, or some other factor may be 
effective. The fact that the toxicity of lA, iB, iC, and 2 A when 
undiluted was not reduced by neutralization with sodium hydrate 
seems to emphasize further the presence of some other factor. It 
is possible that the osmotic pressure of such a concentrated solu- 
tion was high enough to cause injury, although it has been shown 
elsewhere (20) that this is not the cause of the toxicity of the very 
dilute solutions. Antagonistic action on permeability might also 



i9i6] RIGG—SOIL TOXINS 307 

be a possible factor in the lowering of toxicity on the addition of 
sodium hydrate. 

The fact that the toxicity of solution lA was not destroyed by 
the aeration here reported does not necessarily mean that the 
toxicity cannot be removed by oxidation. 

The removal of the toxicity from solution lA by shaking it 
with animal charcoal is probably to be explained as an absorption 
phenomenon. 

The only importance attaching to the toxicity of the soils 
(preparations iD, 2D, and 3D) is that not all of the water-soluble 
toxic materials had been washed out of them by the treatrnents 
to- which they had been subjected. 

The products of the decay of Nymphaea rhizomes are toxic, not 
only to SagiUaria and Tradescantia , but also to agricultural plants. 
Apparently the toxicity of solution lA to tomato, alfalfa, and corn 
is in the order named. 

While the box used for transferring the sterile seeds to sterile 
flask cultures is fairly efficient, the method is somewhat slow and 
tedious, and it is believed that fairly good results may be obtained 
without its use. It seems very desirable to extend our knowledge 
of the growth of seed plants in the absence of other organisms. 

In the one case mentioned, the toxicity of the products of this 
rhizome to corn seemed to be independent of the presence of organ- 
isms, either at the time of dissolving the toxin from the rhizome, or 
at the time of its action on the growing plants. 

The fact that the toxicity of solution lA, even when undiluted, 
can be removed by precipitation with ammonium sulphate seems 
to suggest that the presence of colloidal matter may be a consider- 
able factor in toxicity of that solution. 

An alkaloid similar to nupharin is reported by Wehmer (28) in 
the rhizome of N. alba. He also reports fat and organic acids. 
These also represent possible factors in the toxicity. It seems 
possible that the toxicity may be due partly to products formed 
during decay and partly to products merely released by this decay. 

The toxicity of the water extract of the ash is possibly accounted 
for on the basis of the presence of one or more basic substances. 
Wehmer states that the rhizome of Nymphaea alba contains 



3o8 BOTANICAL GAZETTE [apeil 

9.86 per cent of ash with the following composition: CaO, 8.2 
per cent; CI, 15.2 per cent; NaaO, 48.47 per cent; K2O, q.86 
per cent; P2OS, 14.36 per cent. The presence of sodium hydroxide 
or of sodium carbonate in the water solution of this ash seems 
probable. 

There seemed to be 5 possibiHties as to the cause of the toxicity 
of the products of the decay of this rhizome: (i) the presence of 
the organisms; (2) toxicity due to ionization; (3) the presence of 
toxic molecules; (4) high osmotic pressure of the solutions; and 
(5) low surface tension of the solutions. Of these, (i) seems to 
be practically eliminated by the work here reported. Apparently 
the presence of organisms, either at the time of the formation of 
the solution or at the time of their action on growing plants, is 
not a condition necessary for their toxic action. Work elsewhere 
reported (20) disposes of (4) and (5). This leaves ionization and 
toxic molecules as probable causes of the toxicity. The relative 
importance of these two is not fully determined by the work here 
reported. It is evident, however, that the entire toxicity cannot 
be ascribed to one substance. If we should suppose that the 
toxicity of all of the solutions obtained from this rhizome was due 
to only one substance, it would have to have the following prop- 
erties: (i) soluble in water; (2) soluble in ether; (3) volatile at 
40° C, 20 mm. pressure; (4) stable at 250° C, 15 lbs. pressure; 
(5) absorbed by animal charcoal; and (6) precipitation by am- 
monium sulphate. 

It seems probable that there are at least 3 classes of substances 
here that ar^ somev/hat toxic: (i) colloids, (2) very volatile sub- 
stances, and (3) certain bases. 

Summary 

1. The products of the decay of Nymphaea rhizomes are toxic 
to Tradescantia cuttings, and to tomato, alfalfa, and corn, even in 
very dilute solutions. 

2. All of the solutions prepared except the one from the ash 
were acid, but the amount of acidity was not proportional to the 
degree of toxicity. 



iQi6] RIGG—SOIL TOXINS 309 

3. The toxicity of the very dilute solutions (but not of the strong 
solutions) was nearly destroyed by neutralization with sodium 
hydrate. 

4. The toxicity of solutions resulting directly from the decay 
of the rhizome is destroyed by shaking them with animal charcoal. 

5. The water extract of the fresh rhizome made under 15 lb. 
pressure in an autoclave is toxic to growing plants. 

6. The rhizome and the products of its decay contain some 
ether-soluble toxic material. 

7. Some of the toxic material in the products of the decay of 
these rhizomes is volatile at 40° C. 

8. The toxicity of even concentrated solutions resulting directly 
from the decay of these rhizomes can be removed by precipitation 
with ammonium sulphate. 

9. The water extract of the ash from these rhizomes is basic 
and toxic. 

10. The products of the decay of potatoes, of turnips, and of the 
rhizomes of Castalia odorata and Typha latifolia are also toxic to 
Tradescantia, but slightly less so than those of Nymphaea. 

University of Washington 
Seattle, Wash. 

LITERATURE CITED 

1. Archichowsky, v., Die Wirkung der Giftstaffe verschiedener Konzen- 
trationen auf die Sammen. Biochem. Zeit. 50:233-244. 1913. 

2. Bauman, a., and Gully, E., Uber die freien Humussauren des Hochmoors. 
Mitt. K. Bayr. Moorkulturanst. 4:31-56. 1910; see also Science N.S. 
40:492. 1914. 

3. Bottomly, W. B., The significance of certain food substances for plant 
growth. Ann. Botany 28:531-540. 1914. 

4. Brown, A. J., The selective permeability of the covering of the seeds of 
Hordeimi vulgare. Proc. Roy. Soc. 816:82-93. 1909. 

5. CzAPEK, F., Tiber eine Methode zur direkten bestimmung der Oberblachen- 
spannung der Plasmahaut, von Pflanzenzellen. Jena. 191 1. 

6. Dachnowski, a.. Bog toxins and their effect upon soils. Box. Gaz. 

47: 389-405- 1909- 

7. , Physiological properties of bog water. Box. Gaz. 39:348-355. 1905. 

8. Dandeno, L. B., Mutual interaction of plant roots. Report Mich. Acad. 
Sci. 11:24-25. 1909. 

9. Exper. Sta. Record 15:780. 1903-1904. 



3IO BOTANICAL GAZETTE [april 

10. Fitting, H., Die Wassersorgung und die osmotischen Druckverhaltnisse 
der Wiistenpflanzen. Zeitsch. Bot. 3:209-275. igii. 

11. Harrison, F. C, and Barlow, B., The nodule organization of the Legu- 
minosae. Bakt. Centralbl. 19:264-272, 426-441. 1907. 

iia. Jp:nsen, G. H., Toxic limits and stimulation effects of some salts and 
poisons on wheat. Bot. Gaz. 43:11-44. figs. 34. 1907. 

12. Livingston, B. E., Studies on the properties of an unproductive soil. 
Bull. 28, Bur. of Soils, U.S. Dep. Agric. 1905. 

13. , Further studies on the properties of unproductive soils. Bull. 

36, Bur. of Soils, U.S. Dep. Agric. 1907. 

14. , Physical properties of bog water. Bot. Gaz. 37:383-385. 1904. 

15. Lyon, T. L., and Bizzell, J. A., Is there a mutual stimulation of plants 
through root influence? Jour. Amer. Soc. Agron. 5:38-44. 1913. 

16. MoiLLARD, M., Sur la secretion par les racines de substances toxiques pour 
la plantes (note preliminaire). Bull. Soc. Bot. France. 1914. 

17. Nikitinsky, J., Beeinflussung der Entwickelung einiger Schimmelpilze 
durch ihre Stoffwechselprodukte. Jahrb. Wiss. Bot. 40:1-67. 1904. 

18. Rahn, O., liber den Einfluss der Stoffwechselprokte auf das Wachstum 
der Bakterien. Centralbl. Bakt. II. 16:417-429, 609-617. 1906. 

19. RiGG, G. B., The effect of some Puget Sound bog waters on the root hairs 
of Tradescantia. Bot. Gaz. 55:314-326. 1913. 

20. RiGG, G. B., Trumbull, H. L., and Lincoln, Mattie, Some physical 
properties of certain toxic solutions. To be published in the Botanical 
Gazette. 

21. ScHREiNER, O., The organic constituents of soils. Science 36:577-587. 
1912. 

22. ScHROEDER, H., tjber die Einwirkung von Silbernitrat auf die Klimfahig- 
keit von Getreidkornern. Biol. Centralbl. 35:8-24. 1915. 

23. Sherff, E. E., The vegetation of Skokie marsh, with special reference 
to subterranean organs and their interrelationships. Bot. Gaz. 53:415- 
435. 1912. 

24. Skene, MacGregor, The acidity of sphagnum and its relation to chalk 
and mineral salts. Ann. Botany 29:65-87. 1915. 

25. Skinner, J. J., Illustration of the effect of previous vegetation on a follow- 
ing crop. Plant World 16:342-346. 1913. 

26. Sullivan, M. X., The passage of nucleic acid from plant to medium. 
Science 39:958. 1914. 

27. Wehmer, C, Kleinere mykologische Mitteilungen. Centralbl. Bakt. 
II. 3:102-108. 1897. ' 

28. , Die Pflanzenstoffe. Jena. 1911. 

29. Wieler, a., Pflanzenwachstum und Kalkmangel im Boden. 8vo. pp. 
vii+23S. fi,gs. 43. 1912. 

30. Wilson, J. K., and Harding, H. A., Method of keeping bacteria from 
growing plants. Science 33:544-545. 1911. 



LIBRARY OF CONGRESS 



002 781 720 8 # 



